Identification of a nucleo-cytoplasmic ionic pathway by osmotic shock in isolated mouse liver nuclei

Summary The observation that the nuclear envelope outer mem brane contains ion channels raises the question of whether these conductances communicate between the cytosol and the nuclear envelope cisternae or between the cytosol and the cytoplasm. Failure to detect large, nonselective holes using the patch-clamp technique has led to the speculation that ion channels and nuclear pores are in fact the same. In this paper we present evidence that the ionic channel, recorded in isolated liver nuclei with the patch-clamp configura tion of “nucleus-attached,” spans the double membrane of the envelope, providing a direct contact between nucleoplasm and cytoplasm.     

Examination of DNA sequences undergoing chromatin conformation changes at a variegating breakpoint in Drosophila melanogaster

Position effect variegation in Drosophila melanogaster is associated with the inability of certain genes to be correctly expressed in a proportion of cells, giving a mosaic phenotype. The lack of expression is thought to be due to alterations in the gene's chromatin structure due to its proximity to a region of heterochromatin. Because of the difficulties involved, there is little biochemical data to support the intuitively appealing model of heterochromatin spreading used to explain this phenomenon.Differences in restriction fragment length were used to distinguish DNA regions from either normal (non-position affected) or rearranged (position affected) chromosomes so as to examine possible changes in gene copy number and the effects of endogenous nucleases. DNA sequences at the breakpoint of In (1)w ^m4, which variegates for the white gene, were assayed under conditions where the chromatin conformation was altered using second site modifier mutations (Su(var) or En(var)). No change in the DNA sequerice copy number was observed at either chromosome breakpoint, relative to wild type, when either suppressor or enhancer mutations were present. Therefore copy number change, through differential polyploidization or somatic gene loss, is not affected by Su(var) or En(var) induced changes in the chromatin conformation.Initial experiments showed a gross difference in the sensitivity of DNA to endogenous nucleases that appeared associated with Su(var) and En(var) mutations. En(var) mutation bearing samples appeared delayed in the digestion, relative to Su(var). This differential sensitivity seemed to be genome-wide as there was no detectable difference between either breakpoint of In(1)w ^m4 or the sequences on the homologous w ^- chromosome. However, after isogenizing the genetic background, the previously noted difference between the Su(var) and En(var) mutations was eliminated. In studies dealing with nuclease digestion of chromatin, the isogenization of genetic background is essential before meaningful comparisons can be made.     

The effects of calcium deficiency on Cucurbita pepo L. hypocotyl cells: A 31P nuclear-magnetic-resonance study

Calcium deficiency in zucchini (Cucurbita pepo L.) is associated with reduced growth and a reduced ability to transport auxin (Allan and Rubery, 1991, Planta 183, 604–612). An investigation of the effects of calcium-deficiency on zucchini hypocotyl cells was made using weak-acid uptake and ³¹P-nuclear-magneticresonance (³¹P-NMR) spectroscopy in vivo and in tissue extracts. Calcium-deficient tissue had the same cytoplasmic and vacuolar pHs as normal tissue when extracellular pH was near neutral. At acidic external pH the vacuolar pH was lower in deficient tissue. Adenine nucleotides were present predominantly as ATP in both control and calcium-deficient tissues. Addition of calcium to calcium-deficient tissue, under conditions which cause recovery of auxin transport induced no changes in the ³¹P-NMR spectra of deficient tissue. The content of mobile, phosphorylated metabolites was reduced in calcium-deficient tissue in comparison to control tissue. However, a substantial increase in the content of phosphorylcholine occurs in calcium-deficient tissues compared with controls; this may reflect changes in lipid turnover in calcium-stressed cells. We wish to thank Drs. Terry Moore and Jamie Vandenberg for technical assistance and Professor Peter Morris for providing the gated oxygen device. A.C.A. thanks the Cambridge Commonwealth Trust for a Prince of Wales Scholarship and the O.R.S. Awards Scheme for an award.     

Morphology and molecular phylogeny of the marine diatom genus Nagumoea (Bacillariophyceae) from Japan

The canal-bearing diatom genus Nagumoea, described based on only morphological evidence, was tentatively assigned to the order Bacillariales, although its phylogenetic position remained unclear. Because three isolates of Nagumoea (SK002, SK024 and SK053) were successfully established from Japanese coasts, we performed their morphological observations and molecular phylogenetic analyses to discuss the phylogeny and taxonomic position of this genus. Strains SK002 and SK024 were identified as Nagumoea africana, whereas SK053 conformed with Nagumoea serrata. There was high interspecific divergence between N. africana and N. serrata in the rbcL sequences (8.03–8.17%), indicating their distinctness. Furthermore, intraspecific variations were detected within N. africana (2.35%) in the rbcL, implying its cryptic diversity. The maximum likelihood and Bayesian phylogenetic trees inferred from the plastid rbcL, psbC and nuclear 18S rDNA genes recovered Nagumoea as monophyletic with strong statistical support and embedded within an unresolved, poorly supported lineage containing Achnanthes, Craspedostauros, Staurotropis and Undatella in the canal-bearing order Bacillariales (= the family Bacillariaceae). Although the constrained tree based on the monophyly of Nagumoea and the other canal-bearing clade (Surirellales and Rhopalodiales) was statistically rejected by the topology tests, the phylogenetic position of Nagumoea with other Bacillarialean members remains equivocal. The possession of two plastids positioned fore and aft, observed in the present study, and lack of keel, typical of the Bacillariales, indicate the possibility of Nagumoea being part of the ingroup of the Bacillariales or its closely related outgroup.     

An SAR sequence containing 395 bp DNA fragment mediates enhanced,gene-dosage-correlated expression of a chimaeric heat shock gene in transgenic tobacco plants

A 395 bp fragment located downstream from the soybean heat shock geneGmhsp 17.6-L exhibits several characteristics of scaffold attachment region (SAR) sequences. It contains matrix consensus elements, a topoisomerase II binding sequence and it associates with the isolated nuclear scaffold of soybeanin vitro. Chimaeric genes containing the SAR_L fragment either at one side (5 or 3) or at both sides of a heat shock promoter-regulated -glucuronidase reporter gene were constructed. A five-to nine-fold increase of heat-inducible -glucuronidase activity was observed in transgenic tobacco plants containing constructs with SAR_L fragments either at both sides or with at least one SAR_L copy located upstream from the reporter gene. The gene copy number is positively correlated with the level of heat-inducible reporter gene activity in these. plants but positional effects are not entirely eliminated. Thus, SAR sequences may potentially be used to increase gene expression, via as yet unknown mechanisms, and to reduce adverse effects on the expression of multiple gene copies in transgenic plants.     

Fatty acyl-CoA binding activity of the nuclear thyroid hormone receptor

Long-chain fatty acids and their acyl-CoA esters are potent inhibitors of nuclear thyroid hormone (T₃) receptor in vitro. In the present study, we obtained evidence for acyl-CoA binding activity in the nuclear extract from rat liver. The activity sedimented at a position (3.5 S) identical with that of the T₃ receptor, and the two activities sedimented together. Similarly, they coeluted on DEAE-Sephadex. After partial purification of the receptor, it was again inhibited strongly by acyl-CoAs. Heat stability and a partial trypsin digestion of the receptor both suggested that the action site of oleoyl-CoA overlapped the T₃-binding domain of the receptor. In addition, thyroid hormone receptor β1, synthesized in vitro, bound oleoyl-CoA specifically and its T₃-binding activity was inhibited. The dissociation constant for oleoyl-CoA binding to the partially purified receptor was 1.2 × 10^?7 M. This value as well as its molecular size distinguished the nuclear binding sites from the cytoplasmic fatty acid/acyl-CoA binding proteins. Oleoyl-CoA had no effect on the glucocorticoid receptor, another member of the nuclear hormone-receptor superfamily. From these results, we propose that thyroid hormone receptor is a specific acyl-CoA binding protein of the cell nucleus.     

Fluorescent in situ hybridization of the telomere repeat sequence in hamster sperm nuclear structures

The flat, hooked-shaped architecture of the hamster sperm nucleus makes this an excellent model for in situ hybridization studies of the three dimensional structure of the genome. We have examined the structure of the telomere repeat sequence (TTAGGG)_n with respect to the various nuclear structures present in hamster spermatozoa, using fluorescent in situ hybridization. In fully condensed, mature sperm nuclei, the telomere sequences appeared as discrete spots of various sizes interspersed throughout the volume of the nuclei. While the pattern of these signals was non-random, it varied significantly in different nuclei. These discrete telomere foci were seen to gradually lengthen into linear, beaded signals as sperm nuclei were decondensed, in vitro, and were not associated with the nuclear annulus. We also examined the relationship of telomeres to the sperm nuclear matrix, a residual nuclear structure that retains the original size and shape of the nucleus. In these structures the DNA extends beyond the perimeter of the nucleus to form a halo around it, representing the arrangement of the chromosomal DNA into loop domains attached at their bases to the nuclear matrix. Telomere signals in these structures were also linear and equal in length to those of the decondensed nuclei, and each signal represented part of a single DNA loop domain. The telomeres were attached at one end to the nuclear matrix and extended into the halo. Sperm nuclear matrices treated with Eco RI retained the telomere signals. These data support sperm DNA packaging models in which DNA is coiled into discrete foci, rather than spread out linearly along the length of the sperm nucleus.